PRR lecture, Minerva edition (some graphics and other items removed for copyright and clarity, will be explained in lecture) Prof Ian Sabroe

Covering How we detect microorganisms and viruses The need to detect and respond to molecular ‘patterns’ Receptors on the cell surface and cell membranes: TLRs Receptors inside the cell: NLRs, RLRs

The problem Mr J was well until two days ago. Suddenly he has fever, increased breathlessness, sputum production. What will determine if he lives or dies?

Immunisation Development of immunological memory is powerful and protective but takes weeks… Vaccination/exposure, antigen presentation to T cells, first production of IgM, establishment of high specificity IgG, production of large amounts of IgGs from expanded B cells… If pneumonia, you’re already dead

To handle pathogens we need a rapid response ‘Innate’ immunity: present since birth, no requirement for memory (except an evolutionary one) Neutropaenic sepsis after chemotherapy/BMT

Handling of pathogens Bacteria, fungi: phagocytosis and killing Viruses: cellular shut-down, self-sacrifice, cellular resistance

The problem Immunity can take a long time but infections arrive quickly and unpredictably We live in continual relationship with pathogens and commensals. How many times might you have died today? Infection is often associated with injury, and damage and danger are important concepts

More problems Adaptive immunological memory is highly specific, cross-reacting or self-targeting specificity is a problem. There are a LOT of bacterial species and viruses out there. How do we tell bad from good at a moment’s notice? Only vertebrates have an adaptive system; there must be other effective immune systems.

Charles Janeway 1943-2003 Incredibly for the most ancient part of our immune system, innate immunity and PRR concepts first proposed by Charlie Janeway, 1989 Self-non-self discrimination by recognition of unchanging patterns of microbes

What is pattern recognition?

Patterns Limited characteristics Gram +ve/-ve dsRNA CpG motifs

History of the most famous PRRs

Drosophila Toll

C3H/HeJ: mammals have Toll as well Can’t see LPS/endotoxin Can’t respond to Gram- negative bacteria Science, 1998

The PRR family Secreted and circulating PRRs Cell-associated PRRs (more traditional receptors)

Secreted and circulating PRRs Antimicrobial peptides secreted in lining fluids, from epithelia, and phagocytes. Defensins, cathelicidin Lectins and collectins: carbohydrate-containing proteins that bind carbohydrates or lipids in microbe walls. Activate complement, improve phagocytosis. Mannose binding lectin (deficiency syndromes), surfactant proteins A and D

Secreted and circulating PRRs (2) Pentraxins. Proteins like CRP, which have some antimicrobial actions, can react with the C protein of pneumococci, activate complement, and promote phagocytosis.

Cell-associated PRRs Receptors that are present on the cell membrane or in the cytosol of the cells. Recognise a broad range of molecular patterns. TLRs are the main family

TLRs TLR Ligand (exogenous) Ligand (endogenous) TLR1/2 Gram positive lipopeptides TLR3 Double-stranded RNA mRNA TLR4 LPS, pneumolysin, viral proteins Heat shock proteins, HMGB1, hyaluronan, fibrinogen TLR5 Flagellin TLR2/6 TLR7 Single-stranded RNA RNA? TLR8 TLR9 CpG DNA DNA, mitochondrial DNA? (context may matter)

The special case of TLR4 pg amounts of LPS can signal As little as 10 molecules/cell Polymorphisms in TLR4 affect endotoxin responsiveness, associated with human plagues etc?

What do TLRs do?

Other membrane-bound PRRs Family of receptors that may participate in pathogen recognition and particularly in pathogen phagocytosis: Mannose receptor on macrophages (fungi). Dectin-1. Widespread on phagocytes, helps recognise beta glucans in fungal walls. Scavenger receptors on macrophages.

Not all pathogens are extracellular Viruses multiply in the cytoplasm of cells Bacteria such as Salmonella burst out from the phagolysosome and multiply in the cytoplasm of macrophages

NLRs Named after first members of the family, NODs, Nod-like receptors. Rapidly expanding family of another 22 human proteins that detect intracellular microbial pathogens. Detection of peptidoglycan, muramyl dipeptide, etc. Best known are NOD1, NOD2, NLRP3

NOD2 Widespread expression Recognises muramyl dipeptide (MDP), a breakdown product of peptidoglycan Activates inflammatory signalling pathways Non-functioning mutations: Crohn’s disease Hyperfunctioning mutations: Blau syndrome (rare, chronic granulomatous inflammation of skin, eyes and joints)

RLRs Named after first members of the family: Rig-I. Rig- like helicases = RLRs Best known are RIG-I and MDA5, who’s roles are to detect intracellular double-stranded viral RNA and DNA. They couple effectively to activation of interferon production, enabling an antiviral response NOD2 can also activate anti-viral signalling

So far… Recognition of microbes and viruses depends on seeing ancient, conserved features of them: pattern recognition. Families of receptors exist to detect these in fluids, cell surfaces and compartments, and intracellularly Obvious roles in pathogen responses Additional roles in homeostasis and damage recognition

Homeostasis Blood neutrophil numbers may be dependent upon TLR4 signalling, independent of LPS in homeostasis. Induction of endotoxin tolerance in the newborn gut. Maturation of the normal immune system. Maintaining a balance with commensal organisms.

Polly Matzinger Bunny girl, waitress, jazz musician, dog trainer. And rather a good scientist. Damage hypothesis - hydrophobicity, dynamic interactions.

Damage recognition TLRs also adapted to recognise a range of endogenous damage molecules, which may share characteristics of hydrophobicity. Appearance of host molecules in unfamiliar contexts can activate TLRs. TLR signalling by cellular damage products activates immunity to initiate tissue repair and perhaps enhance local antimicrobial signalling.

PRRs in adaptive immunity Activation of TLRs and other PRRs drives cytokine production by antigen-presenting cells that can increase the likelihood of successful T cell activation. TLR4 agonists already used as vaccine adjuvants. TLR7/8/9 adjuvants in development.

PRRs and disease Recognition of host molecules in autoimmune disease Failure to recognise pathogens or increased inflammatory responses Atherosclerosis, arthritis, COPD, etc.

Translation to therapy Enhance TLR signalling: improve immunity, adjuvants Inhibit TLR signalling: sepsis syndromes Modify adaptive immune response: bias Th and Treg responses

Summary Recognition of molecular patterns is key to survival and successful establishment of and engagement with commensal microbiome PRRs are highly conserved systems of immunity, evolved to recognise unchanging patterns